A Personalized Neoantigen Vaccine in Combination with Platinum-Based Chemotherapy Induces a T-Cell Response Coinciding with a Complete Response in Endometrial Carcinoma.

Endometrial cancer (EC) is a common gynecological malignancy and the fourth most common malignancy in European and North American women. Amongst EC, the advanced serous, p53-mutated, and pMMR subtypes have the highest risk of relapse despite optimal standard of care therapy. At present, there is no standard of care maintenance treatment to prevent relapse among these high-risk patients. Vaccines are a form of immunotherapy that can potentially increase the immunogenicity of pMMR, serous, and p53-mutated tumors to render them responsive to check point inhibitor-based immunotherapy. We demonstrate, for the first time, the feasibility of generating a personalized dendritic cell vaccine pulsed with peptide neoantigens in a patient with pMMR, p53-mutated, and serous endometrial adenocarcinoma (SEC). The personalized vaccine was administered in combination with systemic chemotherapy to treat an inoperable metastatic recurrence. This treatment association demonstrated the safety and immunogenicity of the personalized dendritic cell vaccine. Interestingly, a complete oncological response was obtained with respect to both radiological assessment and the tumor marker CA-125.

Unsupervised Analysis of Flow Cytometry Data in a Clinical Setting Captures Cell Diversity and Allows Population Discovery.

Data obtained with cytometry are increasingly complex and their interrogation impacts the type and quality of knowledge gained. Conventional supervised analyses are limited to pre-defined cell populations and do not exploit the full potential of data. Here, in the context of a clinical trial of cancer patients treated with radiotherapy, we performed longitudinal flow cytometry analyses to identify multiple distinct cell populations in circulating whole blood. We cross-compared the results from state-of-the-art recommended supervised analyses with results from MegaClust, a high-performance data-driven clustering algorithm allowing fast and robust identification of cell-type populations. Ten distinct cell populations were accurately identified by supervised analyses, including main T, B, dendritic cell (DC), natural killer (NK) and monocytes subsets. While all ten subsets were also identified with MegaClust, additional cell populations were revealed (e.g. CD4+HLA-DR+ and NKT-like subsets), and DC profiling was enriched by the assignment of additional subset-specific markers. Comparison between transcriptomic profiles of purified DC populations and publicly available datasets confirmed the accuracy of the unsupervised clustering algorithm and demonstrated its potential to identify rare and scarcely described cell subsets. Our observations show that data-driven analyses of cytometry data significantly enrich the amount and quality of knowledge gained, representing an important step in refining the characterization of immune responses.

Personalized cancer vaccine strategy elicits polyfunctional T cells and demonstrates clinical benefits in ovarian cancer.

T cells are important for controlling ovarian cancer (OC). We previously demonstrated that combinatorial use of a personalized whole-tumor lysate-pulsed dendritic cell vaccine (OCDC), bevacizumab (Bev), and cyclophosphamide (Cy) elicited neoantigen-specific T cells and prolonged OC survival. Here, we hypothesize that adding acetylsalicylic acid (ASA) and low-dose interleukin (IL)-2 would increase the vaccine efficacy in a recurrent advanced OC phase I trial (NCT01132014). By adding ASA and low-dose IL-2 to the OCDC-Bev-Cy combinatorial regimen, we elicited vaccine-specific T-cell responses that positively correlated with patients' prolonged time-to-progression and overall survival. In the ID8 ovarian model, animals receiving the same regimen showed prolonged survival, increased tumor-infiltrating perforin-producing T cells, increased neoantigen-specific CD8+ T cells, and reduced endothelial Fas ligand expression and tumor-infiltrating T-regulatory cells. This combinatorial strategy was efficacious and also highlighted the predictive value of the ID8 model for future ovarian trial development.

Deciphering the Mechanisms of Improved Immunogenicity of Hypochlorous Acid-Treated Antigens in Anti-Cancer Dendritic Cell-Based Vaccines.

Hypochlorous acid (HOCl)-treated whole tumor cell lysates (Ox-L) have been shown to be more immunogenic when used as an antigen source for therapeutic dendritic cell (DC)-based vaccines, improving downstream immune responses both in vitro and in vivo. However, the mechanisms behind the improved immunogenicity are still elusive. To address this question, we conducted a proteomic and immunopeptidomics analyses to map modifications and alterations introduced by HOCl treatment using a human melanoma cell line as a model system. First, we show that one-hour HOCl incubation readily induces extensive protein oxidation, mitochondrial biogenesis, and increased expression of chaperones and antioxidant proteins, all features indicative of an activation of oxidative stress-response pathways. Characterization of the DC proteome after loading with HOCl treated tumor lysate (Ox-L) showed no significant difference compared to loading with untreated whole tumor lysate (FT-L). On the other hand, detailed immunopeptidomic analyses on monocyte-derived DCs (mo-DCs) revealed a great increase in human leukocyte antigen class II (HLA-II) presentation in mo-DCs loaded with Ox-L compared to the FT-L control. Further, 2026 HLA-II ligands uniquely presented on Ox-L-loaded mo-DCs were identified. In comparison, identities and intensities of HLA class I (HLA-I) ligands were overall comparable. We found that HLA-II ligands uniquely presented by DCs loaded with Ox-L were more solvent exposed in the structures of their source proteins, contrary to what has been hypothesized so far. Analyses from a phase I clinical trial showed that vaccinating patients using autologous Ox-L as an antigen source efficiently induces polyfunctional vaccine-specific CD4+ T cell responses. Hence, these results suggest that the increased immunogenicity of Ox-L is, at least in part, due to qualitative and quantitative changes in the HLA-II ligandome, potentially leading to an increased HLA-II dependent stimulation of the T cell compartment (i.e., CD4+ T cell responses). These results further contribute to the development of more effective and immunogenic DC-based vaccines and to the molecular understanding of the mechanism behind HOCl adjuvant properties.

A Phase Ib Study of the Combination of Personalized Autologous Dendritic Cell Vaccine, Aspirin, and Standard of Care Adjuvant Chemotherapy Followed by Nivolumab for Resected Pancreatic Adenocarcinoma-A Proof of Antigen Discovery Feasibility in Three Patients.

Despite the promising therapeutic effects of immune checkpoint blockade (ICB), most patients with solid tumors treated with anti-PD-1/PD-L1 monotherapy do not achieve objective responses, with most tumor regressions being partial rather than complete. It is hypothesized that the absence of pre-existing antitumor immunity and/or the presence of additional tumor immune suppressive factors at the tumor microenvironment are responsible for such therapeutic failures. It is therefore clear that in order to fully exploit the potential of PD-1 blockade therapy, antitumor immune response should be amplified, while tumor immune suppression should be further attenuated. Cancer vaccines may prime patients for treatments with ICB by inducing effective anti-tumor immunity, especially in patients lacking tumor-infiltrating T-cells. These "non-inflamed" non-permissive tumors that are resistant to ICB could be rendered sensitive and transformed into "inflamed" tumor by vaccination. In this article we describe a clinical study where we use pancreatic cancer as a model, and we hypothesize that effective vaccination in pancreatic cancer patients, along with interventions that can reprogram important immunosuppressive factors in the tumor microenvironment, can enhance tumor immune recognition, thus enhancing response to PD-1/PD-L1 blockade. We incorporate into the schedule of standard of care (SOC) chemotherapy adjuvant setting a vaccine platform comprised of autologous dendritic cells loaded with personalized neoantigen peptides (PEP-DC) identified through our own proteo-genomics antigen discovery pipeline. Furthermore, we add nivolumab, an antibody against PD-1, to boost and maintain the vaccine's effect. We also demonstrate the feasibility of identifying personalized neoantigens in three pancreatic ductal adenocarcinoma (PDAC) patients, and we describe their optimal incorporation into long peptides for manufacturing into vaccine products. We finally discuss the advantages as well as the scientific and logistic challenges of such an exploratory vaccine clinical trial, and we highlight its novelty.

Measurement of Mitochondrial Mass and Membrane Potential in Hematopoietic Stem Cells and T-cells by Flow Cytometry.

A fine balance of quiescence, self-renewal, and differentiation is key to preserve the hematopoietic stem cell (HSC) pool and maintain lifelong production of all mature blood cells. In recent years cellular metabolism has emerged as a crucial regulator of HSC function and fate. We have previously demonstrated that modulation of mitochondrial metabolism influences HSC fate. Specifically, by chemically uncoupling the electron transport chain we were able to maintain HSC function in culture conditions that normally induce rapid differentiation. However, limiting HSC numbers often precludes the use of standard assays to measure HSC metabolism and therefore predict their function. Here, we report a simple flow cytometry assay that allows reliable measurement of mitochondrial membrane potential and mitochondrial mass in scarce cells such as HSCs. We discuss the isolation of HSCs from mouse bone marrow and measurement of mitochondrial mass and membrane potential post ex vivo culture. As an example, we show the modulation of these parameters in HSCs via treatment with a metabolic modulator. Moreover, we extend the application of this methodology on human peripheral blood-derived T cells and human tumor infiltrating lymphocytes (TILs), showing dramatic differences in their mitochondrial profiles, possibly reflecting different T cell functionality. We believe this assay can be employed in screenings to identify modulators of mitochondrial metabolism in various cell types in different contexts.

The Candidate Blood-stage Malaria Vaccine P27A Induces a Robust Humoral Response in a Fast Track to the Field Phase 1 Trial in Exposed and Nonexposed Volunteers.

Background: P27A is an unstructured 104mer synthetic peptide from Plasmodium falciparum trophozoite exported protein 1 (TEX1), the target of human antibodies inhibiting parasite growth. The present project aimed at evaluating the safety and immunogenicity of P27A peptide vaccine in malaria-nonexposed European and malaria-exposed African adults. Methods: This study was designed as a staggered, fast-track, randomized, antigen and adjuvant dose-finding, multicenter phase 1a/1b trial, conducted in Switzerland and Tanzania. P27A antigen (10 or 50 µg), adjuvanted with Alhydrogel or glucopyranosil lipid adjuvant stable emulsion (GLA-SE; 2.5 or 5 µg), or control rabies vaccine (Verorab) were administered intramuscularly to 16 malaria-nonexposed and 40 malaria-exposed subjects on days 0, 28, and 56. Local and systemic adverse events (AEs) as well as humoral and cellular immune responses were assessed after each injection and during the 34-week follow-up. Results: Most AEs were mild to moderate and resolved completely within 48 hours. Systemic AEs were more frequent in the formulation with alum as compared to GLA-SE, whereas local AEs were more frequent after GLA-SE. No serious AEs occurred. Supported by a mixed Th1/Th2 cell-mediated immunity, P27A induced a marked specific antibody response able to recognize TEX1 in infected erythrocytes and to inhibit parasite growth through an antibody-dependent cellular inhibition mechanism. Incidence of AEs and antibody responses were significantly lower in malaria-exposed Tanzanian subjects than in nonexposed European subjects. Conclusions: The candidate vaccine P27A was safe and induced a particularly robust immunogenic response in combination with GLA-SE. This formulation should be considered for future efficacy trials. Clinical Trials Registration: NCT01949909, PACTR201310000683408.

50-Gy Stereotactic Body Radiation Therapy to the Dominant Intraprostatic Nodule: Results From a Phase 1a/b Trial.

PURPOSE: Although localized prostate cancer (PCa) is multifocal, the dominant intraprostatic nodule (DIN) is responsible for disease progression after radiation therapy. PCa expresses antigens that could be recognized by the immune system. We therefore hypothesized that stereotactic dose escalation to the DIN is safe, may increase local control, and may initiate tumor-specific immune responses. PATIENTS AND METHODS: Patients with localized PCa were treated with stereotactic extreme hypofractionated doses of 36.25 Gy in 5 fractions to the whole prostate while simultaneously escalating doses to the magnetic resonance image-visible DIN (45 Gy, 47.5 Gy, and 50 Gy in 5 fractions). The phase 1a part was designed to determine the recommended phase 1b dose in a "3 + 3" cohort-based, dose-escalation design. The primary endpoint was dose-limiting toxicities defined as ≥grade 3 gastrointestinal (GI) or genitourinary (GU) toxicity (or both) by National Cancer Institute Common Terminology Criteria for Adverse Events (version 4) up to 90 days after the first radiation fraction. The secondary endpoints were prostate-specific antigen kinetics, quality of life (QoL), and blood immunologic responses. RESULTS: Nine patients were treated in phase 1a. No dose-limiting toxicities were observed at either level, and therefore the maximum tolerated dose was not reached. Further characterization of tolerability, efficacy, and immunologic outcomes was conducted in the subsequent 11 patients irradiated at the highest dose level (50 Gy) in the phase 1b expansion cohort. Toxicity was 45% and 25% for grades 1 and 2 GU, and 20% and 5% for grades 1 and 2 GI, respectively. No grade 3 or worse toxicity was reported. The average (±standard error of the mean) of the QoL assessments at baseline and at 3-month posttreatment were 0.8 (±0.8) and 3.5 (±1.5) for the bowel (mean difference, 2.7; 95% confidence interval, 0.1-5), and 6.4 (±0.8) and 7.27 (±0.9) for the International Prostate Symptom Score (mean difference, 0.87; 95% confidence interval, 0.3-1.9), respectively. A subset of patients developed antigen-specific immune responses against prostate-specific membrane antigen (n = 2), prostatic acid phosphatase (n = 1), prostate stem cell antigen (n = 4), and prostate-specific antigen (n = 2). CONCLUSIONS: Irradiation of the whole prostate with 36.25 Gy in 5 fractions and dose escalation to 50 Gy to the DIN was tolerable and determined as the recommended phase 1b dose. This treatment has promising antitumor activity, which will be confirmed by the ongoing phase 2 part. Preliminary QoL analysis showed minimal impact in GU, GI, and sexual domains. Stereotactic irradiation induced antigen-specific immune responses in a subset of patients.

Safety and immunogenicity of a chimpanzee adenovirus-vectored Ebola vaccine in healthy adults: a randomised, double-blind, placebo-controlled, dose-finding, phase 1/2a study.

BACKGROUND: The ongoing Ebola outbreak led to accelerated efforts to test vaccine candidates. On the basis of a request by WHO, we aimed to assess the safety and immunogenicity of the monovalent, recombinant, chimpanzee adenovirus type-3 vector-based Ebola Zaire vaccine (ChAd3-EBO-Z). METHODS: We did this randomised, double-blind, placebo-controlled, dose-finding, phase 1/2a trial at the Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland. Participants (aged 18-65 years) were randomly assigned (2:2:1), via two computer-generated randomisation lists for individuals potentially deployed in endemic areas and those not deployed, to receive a single intramuscular dose of high-dose vaccine (5 × 10(10) viral particles), low-dose vaccine (2·5 × 10(10) viral particles), or placebo. Deployed participants were allocated to only the vaccine groups. Group allocation was concealed from non-deployed participants, investigators, and outcome assessors. The safety evaluation was not masked for potentially deployed participants, who were therefore not included in the safety analysis for comparison between the vaccine doses and placebo, but were pooled with the non-deployed group to compare immunogenicity. The main objectives were safety and immunogenicity of ChAd3-EBO-Z. We did analysis by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT02289027. FINDINGS: Between Oct 24, 2014, and June 22, 2015, we randomly assigned 120 participants, of whom 18 (15%) were potentially deployed and 102 (85%) were non-deployed, to receive high-dose vaccine (n=49), low-dose vaccine (n=51), or placebo (n=20). Participants were followed up for 6 months. No vaccine-related serious adverse events were reported. We recorded local adverse events in 30 (75%) of 40 participants in the high-dose group, 33 (79%) of 42 participants in the low-dose group, and five (25%) of 20 participants in the placebo group. Fatigue or malaise was the most common systemic adverse event, reported in 25 (62%) participants in the high-dose group, 25 (60%) participants in the low-dose group, and five (25%) participants in the placebo group, followed by headache, reported in 23 (57%), 25 (60%), and three (15%) participants, respectively. Fever occurred 24 h after injection in 12 (30%) participants in the high-dose group and 11 (26%) participants in the low-dose group versus one (5%) participant in the placebo group. Geometric mean concentrations of IgG antibodies against Ebola glycoprotein peaked on day 28 at 51 µg/mL (95% CI 41·1-63·3) in the high-dose group, 44·9 µg/mL (25·8-56·3) in the low-dose group, and 5·2 µg/mL (3·5-7·6) in the placebo group, with respective response rates of 96% (95% CI 85·7-99·5), 96% (86·5-99·5), and 5% (0·1-24·9). Geometric mean concentrations decreased by day 180 to 25·5 µg/mL (95% CI 20·6-31·5) in the high-dose group, 22·1 µg/mL (19·3-28·6) in the low-dose group, and 3·2 µg/mL (2·4-4·9) in the placebo group. 28 (57%) participants given high-dose vaccine and 31 (61%) participants given low-dose vaccine developed glycoprotein-specific CD4 cell responses, and 33 (67%) and 35 (69%), respectively, developed CD8 responses. INTERPRETATION: ChAd3-EBO-Z was safe and well tolerated, although mild to moderate systemic adverse events were common. A single dose was immunogenic in almost all vaccine recipients. Antibody responses were still significantly present at 6 months. There was no significant difference between doses for safety and immunogenicity outcomes. This acceptable safety profile provides a reliable basis to proceed with phase 2 and phase 3 efficacy trials in Africa. FUNDING: Swiss State Secretariat for Education, Research and Innovation (SERI), through the EU Horizon 2020 Research and Innovation Programme.

Safety of human immunisation with a live-attenuated Mycobacterium tuberculosis vaccine: a randomised, double-blind, controlled phase I trial.

BACKGROUND: Tuberculosis remains one of the world's deadliest transmissible diseases despite widespread use of the BCG vaccine. MTBVAC is a new live tuberculosis vaccine based on genetically attenuated Mycobacterium tuberculosis that expresses most antigens present in human isolates of M tuberculosis. We aimed to compare the safety of MTBVAC with BCG in healthy adult volunteers. METHODS: We did this single-centre, randomised, double-blind, controlled phase 1 study at the Centre Hospitalier Universitaire Vaudois (CHUV; Lausanne, Switzerland). Volunteers were eligible for inclusion if they were aged 18-45 years, clinically healthy, HIV-negative and tuberculosis-negative, and had no history of active tuberculosis, chemoprophylaxis for tuberculosis, or BCG vaccination. Volunteers fulfilling the inclusion criteria were randomly assigned to three cohorts in a dose-escalation manner. Randomisation was done centrally by the CHUV Pharmacy and treatments were masked from the study team and volunteers. As participants were recruited within each cohort, they were randomly assigned 3:1 to receive MTBVAC or BCG. Of the participants allocated MTBVAC, those in the first cohort received 5 × 10(3) colony forming units (CFU) MTBVAC, those in the second cohort received 5 × 10(4) CFU MTBVAC, and those in the third cohort received 5 × 10(5) CFU MTBVAC. In all cohorts, participants assigned to receive BCG were given 5 × 10(5) CFU BCG. Each participant received a single intradermal injection of their assigned vaccine in 0·1 mL sterile water in their non-dominant arm. The primary outcome was safety in all vaccinated participants. Secondary outcomes included whole blood cell-mediated immune response to live MTBVAC and BCG, and interferon γ release assays (IGRA) of peripheral blood mononuclear cells. This trial is registered with ClinicalTrials.gov, number NCT02013245. FINDINGS: Between Jan 23, 2013, and Nov 6, 2013, we enrolled 36 volunteers into three cohorts, each of which consisted of nine participants who received MTBVAC and three who received BCG. 34 volunteers completed the trial. The safety of vaccination with MTBVAC at all doses was similar to that of BCG, and vaccination did not induce any serious adverse events. All individuals were IGRA negative at the end of follow-up (day 210). After whole blood stimulation with live MTBVAC or BCG, MTBVAC was at least as immunogenic as BCG. At the same dose as BCG (5×10(5) CFU), although no statistical significance could be achieved, there were more responders in the MTBVAC group than in the BCG group, with a greater frequency of polyfunctional CD4+ central memory T cells. INTERPRETATION: To our knowledge, MTBVAC is the first live-attenuated M tuberculosis vaccine to reach clinical assessment, showing similar safety to BCG. MTBVAC seemed to be at least as immunogenic as BCG, but the study was not powered to investigate this outcome. Further plans to use more immunogenicity endpoints in a larger number of volunteers (adults and adolescents) are underway, with the aim to thoroughly characterise and potentially distinguish immunogenicity between MTBVAC and BCG in tuberculosis-endemic countries. Combined with an excellent safety profile, these data support advanced clinical development in high-burden tuberculosis endemic countries. FUNDING: Biofabri and Bill & Melinda Gates Foundation through the TuBerculosis Vaccine Initiative (TBVI).

Safety and immunogenicity of the M72/AS01 candidate tuberculosis vaccine in HIV-infected adults on combination antiretroviral therapy: a phase I/II, randomized trial.

OBJECTIVE: Tuberculosis (TB) is highly prevalent among HIV-infected people, including those receiving combination antiretroviral therapy (cART), necessitating a well tolerated and efficacious TB vaccine for these populations. We evaluated the safety and immunogenicity of the candidate TB vaccine M72/AS01 in adults with well controlled HIV infection on cART. DESIGN: A randomized, observer-blind, controlled trial (NCT00707967). METHODS: HIV-infected adults on cART in Switzerland were randomized 3 : 1 : 1 to receive two doses, 1 month apart, of M72/AS01, AS01 or 0.9% physiological saline (N = 22, N = 8 and N = 7, respectively) and were followed up to 6 months postdose 2 (D210). Individuals with CD4⁺ cell counts below 200 cells/µl were excluded. Adverse events (AEs) including HIV-specific and laboratory safety parameters were recorded. Cell-mediated (ICS) and humoral (ELISA) responses were evaluated before vaccination, 1 month after each dose (D30, D60) and D210. RESULTS: Thirty-seven individuals [interquartile range (IQR) CD4⁺ cell counts at screening: 438-872 cells/µl; undetectable HIV-1 viremia] were enrolled; 73% of individuals reported previous BCG vaccination, 97.3% tested negative for the QuantiFERON-TB assay. For M72/AS01 recipients, no vaccine-related serious AEs or cART-regimen adjustments were recorded, and there were no clinically relevant effects on laboratory safety parameters, HIV-1 viral loads or CD4⁺ cell counts. M72/AS01 was immunogenic, inducing persistent and polyfunctional M72-specific CD4⁺ T-cell responses [medians 0.70% (IQR 0.37-1.07) at D60] and 0.42% (0.24-0.61) at D210, predominantly CD40L⁺IL-2⁺TNF-α⁺, CD40L⁺IL-2⁺ and CD40L⁺IL-2⁺TNF-α⁺IFN-γ⁺]. All M72/AS01 vaccines were seropositive for anti-M72 IgG after second vaccination until study end. CONCLUSION: M72/AS01 was clinically well tolerated and immunogenic in this population, supporting further clinical evaluation in HIV-infected individuals in TB-endemic settings.

Novel birch pollen specific immunotherapy formulation based on contiguous overlapping peptides.

BACKGROUND: Synthetic contiguous overlapping peptides (COPs) may represent an alternative to allergen extracts or recombinant allergens for allergen specific immunotherapy. In combination, COPs encompass the entire allergen sequence, providing all potential T cell epitopes, while preventing IgE conformational epitopes of the native allergen. METHODS: Individual COPs were derived from the sequence of Bet v 1, the major allergen of birch pollen, and its known crystal structure, and designed to avoid IgE binding. Three sets of COPs were tested in vitro in competition ELISA and basophil degranulation assays. Their in vivo reactivity was determined by intraperitoneal challenge in rBet v 1 sensitized mice as well as by skin prick tests in volunteers with allergic rhinoconjunctivitis to birch pollen. RESULTS: The combination, named AllerT, of three COPs selected for undetectable IgE binding in competition assays and for the absence of basophil activation in vitro was unable to induce anaphylaxis in sensitized mice in contrast to rBet v 1. In addition no positive reactivity to AllerT was observed in skin prick tests in human volunteers allergic to birch pollen. In contrast, a second set of COPs, AllerT4-T5 displayed some residual IgE binding in competition ELISA and a weak subliminal reactivity to skin prick testing. CONCLUSIONS: The hypoallergenicity of contiguous overlapping peptides was confirmed by low, if any, IgE binding activity in vitro, by the absence of basophil activation and the absence of in vivo induction of allergic reactions in mouse and human. TRIAL REGISTRATION: ClinicalTrials.gov NCT01719133.

Intragastric and Intranasal Administration of Lactobacillus paracasei NCC2461 Modulates Allergic Airway Inflammation in Mice.

Introduction. Preclinical and clinical evidences for a role of oral probiotics in the management of allergic diseases are emerging. Aim. We aimed at testing the immunomodulatory effects of intranasal versus intragastric administration of Lactobacillus paracasei NCC2461 in a mouse model of allergic airway inflammation and the specificity of different probiotics by comparing L. paracasei NCC2461 to Lactobacillus plantarum NCC1107. Methods. L. paracasei NCC2461 or L. plantarum NCC1107 strains were administered either intragastrically (NCC2461) or intranasally (NCC2461 or NCC1107) to OVA-sensitized mice challenged with OVA aerosols. Inflammatory cell recruitment into BALF, eotaxin and IL-5 production in the lungs were measured. Results. Intranasal L. paracasei NCC2461 efficiently protected sensitized mice upon exposure to OVA aerosols in a dose-dependent manner as compared to control mice. Inflammatory cell number, eotaxin and IL-5 were significantly reduced in BALF. Intranasal supplementation of L. paracasei NCC2461 was more potent than intragastric application in limiting the allergic response and possibly linked to an increase in T regulatory cells in the lungs. Finally, intranasal L. plantarum NCC1107 reduced total and eosinophilic lung inflammation, but increased neutrophilia and macrophages infiltration. Conclusion. A concerted selection of intervention schedule, doses, and administration routes (intranasal versus intragastric) may markedly contribute to modulate airway inflammation in a probiotic strain-specific manner.

Synthetic chemokines directly labeled with a fluorescent dye as tools for studying chemokine and chemokine receptor interactions.

Chemokines constitute a protein family that exhibit a variety of biological activities involved in normal and pathological physiological processes. CCL11 (eotaxin), CCL19 (MIP-3beta), CCL22 (MDC), CXCL11 (I-TAC) and CXCL12 (SDF-1alpha) chemokines, modified with the Alexa Fluor 647 fluorescent dye at specific positions along their sequence, were produced by a chemical route and their biological activities were characterized. In a migration assay, fluorescent chemokines were as biologically active as the unmodified forms. All labeled chemokines specifically stained cell lines transfected with the appropriate human chemokine receptors. The specificity of binding was further established by showing that the unlabeled ligands efficiently competed with the labeled chemokines for binding to their respective receptor. A low molecular weight antagonist of CXCR4 prevented binding of labeled CXCL12 to CXCR4 comparably to a neutralizing anti-CXCR4 antibody. Finally, labeled CCL19 was used for the staining of primary cells, illustrating that this reagent can be used for studying CCR7 expression on different cell types. Together, these results demonstrate that fluorescent synthetic chemokines constitute promising ligands for the development of chemokine receptor-binding assays on intact cells, for applications such as cell-based, high throughput screening, and studies of chemokine receptor expression by primary cells.

Biotinylated synthetic chemokines: their use for the development of nonradioactive whole-cell binding assays.

A chemokine binding assay on whole cells was developed using biotinylated synthetic CCL22 as a model ligand. CCL22 analogues were produced by a chemical route, resulting in > 97% homogeneous and defined polypeptides. First, the 5 biotinylated CCL22 analogues synthesized were captured by agarose-immobilized streptavidin, indicating that the biotin molecules introduced in positions G1, K27, K49, K61, and K66 of CCL22 were accessible for binding. Then, it was established using a migration assay that the biotinylated chemokines were at least as biologically active as the unmodified CCL22 form. Subsequently, the biotinylated chemokines were evaluated in an FACS-based whole-cell binding assay. Surprisingly, only the CCL22 analogue with the biotin in position K66 constituted a suitable staining reagent for CCR4-positive cells. Finally, binding characteristics and reproducibility of the binding assay were outlined for the CCL22 analogue with the biotin in position K66. These results exemplified that biotinylated synthetic chemokines constitute promising ligands for the development of chemokine receptor-binding assays on whole cells, provided the position of the biotin moiety introduced along the sequence is adequately chosen.

Phosphodiesterase 4 inhibitors reduce human dendritic cell inflammatory cytokine production and Th1-polarizing capacity.

Inhibitors of cAMP-specific phosphodiesterase (PDE) 4 have been shown to inhibit inflammatory mediator release and T cell proliferation, and are considered candidate therapies for T(h)1-mediated diseases. However, little is known about how PDE4 inhibitors influence dendritic cells (DC), the cells responsible for the priming of naive T(h) cells. Therefore, we investigated the PDE profile of monocyte-derived DC, and whether PDE4 inhibitors modulate DC cytokine production and T cell-polarizing capacity. We mainly found cAMP-specific PDE4 enzymatic activity in both immature and mature DC. In contrast to monocytes that mainly express PDE4B, we found that PDE4A is the predominant PDE4 subtype present in DC. Immature DC showed reduced ability to produce IL-12p70 and tumor necrosis factor (TNF)-alpha upon lipopolysaccharide or CD40 ligand (CD40L) stimulation in the presence of PDE4 inhibitors, whereas cytokine production upon CD40L stimulation of fully mature DC in the presence of PDE4 inhibitors was not affected. Exposure to PDE4 inhibitors for 2 days during DC maturation did not influence T cell-stimulatory capacity or acquisition of a mature phenotype, but increased the expression of the chemokine receptor CXCR4. Furthermore, DC matured in the presence of PDE4 inhibitors showed reduced capacity to produce IL-12p70 and TNF-alpha upon subsequent CD40L stimulation. Using these PDE4 inhibitor-matured DC to stimulate naive T cells resulted in a reduction of IFN-gamma-producing (T(h)1) cells. These findings indicate that PDE4 inhibitors can affect T cell responses by acting at the DC level and may increase our understanding of the therapeutic implication of PDE4 inhibitors for T(h)1-mediated disorders.